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Te Tari Moromoroiti me te Ārai Mate

Publication of the month


January 2017

Inclusion of BLIMP-1+ effector regulatory T cells improves the Immunoscore in a cohort of New Zealand colorectal cancer patients: a pilot study

Kirsten A. Ward-Hartstonge, John L. McCall, Timothy R. McCulloch, Ann-Kristin Kamps, Adam Girardin, Erika Cretney, Fran M. Munro, Roslyn A. Kemp

Cancer Immunology, Immunotherapy, January 2017. DOI: 10.1007/s00262-016-1951-1

Abstract 

Analysis of tumour-infiltrating T cells in colorectal cancer can predict disease-free survival. The Immunoscore, obtained by quantifying tumour-infiltrating CD3+ and CD8+ T cells, may improve current staging. Effector regulatory T cells are a potently suppressive subset in mice and, while present in human colorectal cancer, their role in patient outcome is unknown. Immunofluorescence was used to analyse immune cell infiltrates in patients with early (stage II) colorectal cancer with (n = 13) and without (n = 19) recurrent disease. CD3 and CD8 were used for the Immunoscore; FOXP3, BLIMP-1 and CD3 to identify effector regulatory T cells. Patients with high Immunoscores had increased disease-free survival compared to patients with low Immunoscores (Log-rank test p < 0.01). Prediction of outcome was further improved by stratifying patients with a low Immunoscore according to CD3+FOXP3+BLIMP-1+ cell infiltration at the invasive margin. Patients with a low Immunoscore and high infiltrate of CD3+FOXP3+BLIMP-1+ cells tended to have better disease-free survival than patients with low Immunoscore and low infiltrate of CD3+FOXP3+BLIMP-1+ cells. Patients with a high Immunoscore had better disease-free survival than patients with a low Immunoscore and low infiltrate of CD3+ FOXP3+ BLIMP-1+ cells (Log-rank test p < 0.001). These results indicate that tumour infiltration with effector regulatory T cells improves the prognostic value of the Immunoscore and implies that these cells may play a role in colorectal cancer patient outcome.

The people involved

Kirsten Ward-Hartstonge is PhD student in the Kemp Lab, and produced the work along with the help of her supervisor, Associate Professor Roslyn Kemp, and fellow students. Collaborators came from the Department of Surgical Sciences at the University of Otago, as well as The Walter and Eliza Hall Institute of Medical Research and the University of Melbourne, Australia.

Read the full paper online


December 2016

Differential growth of bowel commensal Bacteroides species on plant xylans of differing structural complexity

Manuela Centanni, Jennifer C. Hutchison, Susan M. Carnachan, Alison M. Daines, William J. Kelly, Gerald W. Tannock, Ian M. Sims

Carbohydrate Polymers, Volume 157, 10 February 2017, Pages 1374–1382

Abstract

Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.

The people involved

Dr Manuela Centanni is a Postdoctoral Fellow in Professor Gerald Tannock's lab (which is also affiliated with the research theme Microbiome Otago). Collaborative partners included Victoria University of Wellington's Ferrier Research Institute, Donvis Limited and the Riddet Insitute (both situated in Palmerston North).

Read the full paper online

December 2016: student publication of the month

Phylogenetic and functional potential links pH and N2O emissions in pasture soils

M. d. Sainur Samad, Ambarish Biswas, Lars R. Bakken, Timothy J. Clough, Cecile A. M. de Klein, Karl G. Richards, Gary J. Lanigan, and Sergio E. Morales

Scientific Reports 2016; 6: 35990. DOI: 10.1038/srep35990

Abstract

Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir & nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.

The people involved

The lead author, M. d. Sainur Samad, is a PhD student in Sergio Morales' lab along with Postdoctoral Fellow and departmental Bioinformatician Ambarish Biswas. Collaborators are based in Norway and Ireland, and more locally at AgResearch Invermay and the Lincoln University.

Read the full paper online


November 2016

Quorum sensing controls adaptive immunity through the regulation of multiple CRISPR-Cas systems

Adrian G. Patterson, Simon A. Jackson, Corinda Taylor, Gary B. Evans, George P.C. Salmond, Rita Przybilski, Raymond H.J. Staals, Peter C. Fineran

Molecular Cell, November 2016, DOI: http://dx.doi.org/10.1016/j.molcel.2016.11.012

Summary

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

The people involved

The project was led by the Fineran Lab (Department of Microbiology and Immunology, University of Otago) with contributions from Gary Evans (Ferrier Research Institute, Victoria University of Wellington) and George Salmond (Department of Biochemistry, University of Cambridge).

fineran group 3

From left to right: Dr Simon Jackson, Dr Raymond Staals, Associate Professor Peter Fineran, Adrian Patterson, Dr Rita Przybilski.

Read the full paper online

Read the media release on the University of Otago website

November 2016: student publication of the month

Functional impairment of infiltrating T cells in human colorectal cancer

Edward S. Taylor, John L. McCall, Adam Girardin, Fran M. Munro, Michael A. Black and Roslyn A. Kemp

OncoImmunology, Volume 5, 2016 - Issue 11

Abstract

T cells play a crucial role in preventing the growth and spread of colorectal cancer (CRC). However, immunotherapies against CRC have only shown limited success, which may be due to lack of understanding about the effect of the local tumor microenvironment (TME) on T cell function. The goal of this study was to determine whether T cells in tumor tissue were functionally impaired compared to T cells in non-tumor bowel (NTB) tissue from the same patients. We showed that T cell populations are affected differently by the TME. In the tumor, T cells produced more IL-17 and less IL-2 per cell than their counterparts from NTB tissue. T cells from tumor tissue also had impaired proliferative ability compared to T cells in NTB tissue. This impairment was not related to the frequency of IL-2 producing T cells or regulatory T cells, but T cells from the TME had a higher co-expression of inhibitory receptors than T cells from NTB. Overall, our data indicate that T cells in tumor tissue are functionally altered by the CRC TME, which is likely due to cell intrinsic factors. The TME is therefore an important consideration in predicting the effect of immune modulatory therapies.

The people involved

Edward Taylor was a PhD student in Dr Roslyn Kemp's lab. The projkect was a collaboration with University of Otago colleagues in the Department of Surgical Sciences and the Department of Biochemistry.

Read the full paper online


October 2016

Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR–Cas system

Raymond H. J. Staals, Simon A. Jackson, Ambarish Biswas, Stan J. J. Brouns, Chris M. Brown & Peter C. Fineran

Nature Communications 7, 12853 (2016), doi:10.1038/ncomms12853

Summary

Just like humans, bacteria are constantly under attack by viruses that try to infect them by injecting viral DNA. CRISPR-Cas systems can remember and destroy invading DNA by storing short, invader-derived, pieces of DNA (called 'spacers') into their genetic memory banks. A previous study demonstrated that that viruses and plasmids, which can avoid recognition by mutating their DNA, will trigger the bacteria's CRISPR-Cas system to respond by quickly acquiring new immunological memories from these mutated threats. This process is called 'primed adaptation'.

In the current study, the team sought to understand how these new memories were selected during the primed adaptation response. They discovered that the acquisition of memories from plasmids not previously encountered was very inefficient in comparison to the mutated plasmids. By studying the order in which these new memories were acquired, the researchers developed a new model for primed adaptation. The data indicated that acquisition of new immunological memory occurs not only during primed adaptation but also as a consequence of direct defence against invading genetic elements that have not mutated to evade recognition by the CRISPR-Cas system.

Abstract

CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition’) is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.

Read the paper online

October 2016: student publication of the month

Structure and function of AmtR in Mycobacterium smegmatis: implications for post-transcriptional regulation of urea metabolism through a small antisense RNA

Michael Petridis, Chelsea Vickers, Jennifer Robson, Joanna L. McKenzie, Magdalena Bereza, Abigail Sharrock, Htin Lin Aung, Vickery L. Arcus and Gregory M. Cook

Journal of Molecular Biology (2016) 428, 4315-4329, http://dx.doi.org/10.1016/j.jmb.2016.09.009

Abstract

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ΔamtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N4 -GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis -encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0 Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis  AmtR interaction with the adenylylated PII protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis -encoded small antisense RNA.

The people involved

The project was a collaboration between the University of Otago's Department of Microbiology and Immunology, the University of Waikato's Department of Biological Sciences and the Maurice Wilkins Centre for Molecular Biodiversity, with funding provided by the Maurice Wilkins Centre and a Marsden Grant from the Royal Society of New Zealand.

Michael Petridis, now a Postdoctoral Fellow in the Cook Lab, undertook this work as a part of a PhD with the Department of Microbiology and Immunology. He was supported by a University of Otago Doctoral Scholarship, the Webster Center for Infectious Diseases and the Otago School of Medical Sciences.

Read the paper online


September 2016

Structures of exopolysaccharides involved in receptor-mediated perception of Mesorhizobium loti in Lotus japonicus

Artur Muszyński, Christian Heiss, Christian T. Hjuler, John T. Sullivan, Simon J. Kelly, Mikkel B. Thygesen, Jens Stougaard, Parastoo Azadi, Russell W. Carlson, and Clive W. Ronson

The Journal of Biological Chemistry, August 2016

Summary

In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Our recent collaborative studies (Kawaharada, Y. et al. (2015) Nature 523, 308–312) showed that M. loti low molecular mass exopolysaccharides are perceived by the L. japonicus receptor-like kinase, EPR3, and serve as signal molecules that regulate infection thread formation and bacterial passage through the plant’s epidermal cell layer during root nodule formation. In the current paper, we characterize in detail the structures of high and low molecular mass EPS isolated from M. loti R7A including the locations of non-stoichiometric O-acetyl substituents. We demonstrate that the repeating unit of EPS polymer consists of highly O-acetylated octasaccharide with acidic branch substituted with an a-d-glucuronic and β-d-riburonic acids. In addition we propose that the low molecular mass octasaccharide EPS that is recognized by EPR3 kinase receptor is the monomer of the biosynthetic repeating oligosaccharide unit of the high molecular mass polymeric EPS. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and lead to a better understanding of the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.

The people involved

This paper is the outcome of a truly global collaboration involving researchers in four institutions and three countries, the United States, Denmark and New Zealand. The work was carried out under the umbrella of the Centre for Carbohydrate Recognition and Signalling (carb.au.dk), a Centre of Excellence supported financially by the Danish National Research Foundation and led by Professor Jens Stougaard at the University of Aarhus. The work was led by Professor Clive Ronson from the University of Otago who provided the biological expertise, and by Dr Artur Muszyński at the Centre for Complex Carbohydrate Research in Athens, Georgia, USA, who led the chemistry work, and they are joint corresponding authors on the paper. Researchers at the University of Copenhagen also made important contributions to the chemical characterization, especially of the low molecular mass EPS. Other contributors from Otago were Simon Kelly who was a PhD student for most of the study and who is currently a postdoctoral fellow at Aarhus, and Dr John Sullivan who is an assistant research fellow in the Ronson lab.

Read the paper online

R7A blueWEB

The authors also supplied the journal cover image. Photo credit: Stefan Eberhard, Christian T. Hjuler, and Artur Muszyński.

SEM (×370) image of freeze-dried extract of exopolysaccharide (EPS) isolated from Mesorhizobium loti strain R7A. This high molecular mass polymer consists of O-acetylated octasaccharide repeating units. A single monomeric O-acetylated octasaccharide, demonstrated in the lowest energy 3D structural model (overlay), is a biosynthetic repeat of EPS and plays a signaling role in receptor-mediated symbiotic infection of Lotus japonicus by M. loti.

September 2016: student publication of the month

Expression of the NS5 (VPg) protein of murine norovirus induces a G1/S phase arrest

Colin Davies, Vernon K. Ward

PLOS One, August 2016

Abstract 

Murine norovirus-1 (MNV-1) is known to subvert host cell division inducing an accumulation of cells in the G0/G1 phase, creating conditions where viral replication is favored. This study identified that NS5 (VPg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to MNV-1 infection. NS5 expression induced an accumulation of cells in the G0/G1 phase in an asynchronous population by inhibiting progression at the G1/S restriction point. Furthermore, NS5 expression resulted in a down-regulation of cyclin A expression in asynchronous cells and inhibited cyclin A expression in cells progressing from G1 to S phase. The activity of NS5 on the host cell cycle occurs through an uncharacterized function. Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G0/G1 phase as identified for wild-type NS5. To the best of our knowledge, this is the first report of a VPg protein manipulating the host cell cycle.

The people involved

Colin Davies recently completed his PhD in the Department of Microbiology, supervised by Professor Vernon Ward.

Read the paper online


August 2016

Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles

Morad-Rémy Muhsin-Sharafaldine, Sarah C. Saunderson, Amy C. Dunn, James M. Faed, Torsten Kleffmann, Alexander D. McLellan

Oncotarget, June 2016 

Summary

The study shows that tiny vesicles released from the surface of tumour cells during chemotherapy contribute to thrombotic events in cancer patients. These disorders of the coagulation system, such as deep vein thrombosis and pulmonary embolism, are the second highest cause of death in cancer patients. The work carried out by lead author Remy Muhsin (PhD student) has identified a new system of pathological coagulation that is a rapid, short circuit version of physiological coagulation

Abstract

Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity. 

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July 2016

Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species

April Pawluk, Raymond H.J. Staals, Corinda Taylor, Bridget N.J. Watson, Senjuti Saha, Peter C. Fineran, Karen L. Maxwell and Alan R. Davidson

Nature Microbiology, vol 1, August 2016

Summary

The authors of this paper have uncovered a new arsenal of anti-CRISPR proteins produced by viruses that enable them to circumvent CRISPR-Cas adaptive immunity in their arms race with bacteria. CRISPR-Cas systems come in many different varieties, and remarkably, one of the newly discovered anti-CRISPR proteins was dual specificity, inhibiting the activity of two types of CRISPR-Cas systems. The apparent widespread distribution of anti-CRISPRs suggest that phages and plasmids can evade bacterial immunity to allow their transfer between bacteria. 

Abstract

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti- CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

The people involved

The project was led from the groups of Alan Davidson and Karen Maxwell from the University of Toronto. Collaborators from Associate Professor Peter Fineran’s group were Postdoctoral Fellow Dr Raymond Staals, Research Assistant Corinda Taylor and PhD student Bridget Watson.

Read the full article


June 2016

Influenza A Virus Dysregulates Host Histone Deacetylase 1 That Inhibits Viral Infection in Lung Epithelial Cells

Prashanth Thevkar Nagesh, Matloob Husain

Journal of Virology, 90(9) May 2016

Summary

Influenza A virus (IAV) continues to significantly impact global public health by causing regular seasonal epidemics, occasional pandemics, and zoonotic outbreaks. IAV is among the successful human viral pathogens that has evolved various strategies to evade host defenses, prevent the development of a universal vaccine, and acquire antiviral drug resistance. A comprehensive knowledge of IAV-host interactions is needed to develop a novel and alternative anti-IAV strategy. Host produces a variety of factors that are able to fight IAV infection by employing various mechanisms. However, the full repertoire of anti-IAV host factors and their antiviral mechanisms has yet to be identified. We have identified here a new host factor, histone deacetylase 1 (HDAC1) that inhibits IAV infection. We demonstrate that HDAC1 is a component of host innate antiviral response against IAV, and IAV undermines HDAC1 to limit its role in antiviral response.

Abstract

Viruses dysregulate the host factors that inhibit virus infection. Here, we demonstrate that human enzyme, histone deacetylase 1 (HDAC1) is a new class of host factor that inhibits influenza A virus (IAV) infection, and IAV dysregulates HDAC1 to efficiently replicate in epithelial cells. A time-dependent decrease in HDAC1 polypeptide level was observed in IAV-infected cells, reducing to<50% by 24 h of infection. A further depletion (97%) of HDAC1 expression by RNA interference increased the IAV growth kinetics, increasing it by>3-fold by 24 h and by>6-fold by 48 h of infection. Conversely, overexpression of HDAC1 decreased the IAV infection by>2-fold. Likewise, a time-dependent decrease in HDAC1 activity, albeit with slightly different kinetics to HDAC1 polypeptide reduction, was observed in infected cells. Nevertheless, a further inhibition of deacetylase activity increased IAV infection in a dose-dependent manner. HDAC1 is an important host deacetylase and, in addition to its role as a transcription repressor, HDAC1 has been lately described as a coactivator of type I interferon response. Consistent with this property, we found that inhibition of deacetylase activity either decreased or abolished the phosphorylation of signal transducer and activator of transcription I (STAT1) and expression of interferon-stimulated genes, IFITM3, ISG15, and viperin in IAV-infected cells. Furthermore, the knockdown of HDAC1 expression in infected cells decreased viperin expression by 58% and, conversely, the overexpression of HDAC1 increased it by 55%, indicating that HDAC1 is a component of IAV-induced host type I interferon antiviral response.

The people involved

This paper is a direct output from PhD thesis research by Prashanth Thevkar Nagesh (Department of Microbiology and Immunology). Dr Matloob Husain is his supervisor.

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May 2016

Gut macrophage phenotype is dependent on the tumor microenvironment in colorectal cancer

Samuel E Norton, Elliott TJ Dunn, John L McCall, Fran Munro and Roslyn A Kemp

Clinical & Translational Immunology (2016) 5, e76; doi:10.1038/cti.2016.21

Abstract

In contrast to many cancers, a high infiltration of macrophages in colorectal cancer (CRC) has been associated with improved prognosis for patients. Cytokines and other stimuli from the tumor microenvironment affect monocyte to macrophage maturation and subsequent phenotype and function. Heterogeneous myeloid populations were identified using a novel flow cytometry panel in both tumor and paired non-tumor bowel (NTB) from CRC patients. The frequency of macrophage subsets with a gut-conditioned phenotype was lower in tumor compared with NTB. We used an in vitro system to show that two of the macrophage populations represented pro-inflammatory and anti-inflammatory phenotypes. Conditioned media that contained high levels of interleukin-6 promoted and maintained an anti-inflammatory phenotype in vitro. This study demonstrates the plasticity and heterogeneity of macrophage subtypes in human CRC, and the feasibility of studying complex populations. Ex vivo experiments demonstrate that macrophage subsets are influenced by the tumor microenvironment.

The people involved

This work was undertaken as part of Sam Norton's PhD research. Dr Roslyn Kemp, Department of Microbiology and Immunology is his supervisor, and Elliott Dunn is a former student of the Kemp Lab. John McCall and Fran Munro are based in the University of Otago's Department of Surgical Sciences.

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April 2016

Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12

Tania L. Slatter, Michelle Wilson, Chingwen Tang, Hamish G. Campbell, Vernon K. Ward, Vivienne L. Young, David Van Ly, Nicholas I. Fleming, Antony W. Braithwaite & Margaret A. Baird

OncoImmunology, 5(3), e1112941. doi: 10.1080/2162402X.2015.1112941

Summary of the work

In this project it was found that the tumour supressor protein p53 plays an important role in helping APC (activated antigen-presenting cells) generate an antitumour response. This demonstrated a completely new role for this protein and could contribute to future immunotherapies.

Abstract

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11cC APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigenspecific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

The people involved

Michelle Wilson is the Department of Microbiology and Immunology's Flow Cytometry Technician and Professor Vernon Ward is a virology researcher. This work was undertake alongside colleagues from the Department of Pathology, the Maurice Wilkins Centre and the University of Sydney.

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March 2016

Digestive-resistant carbohydrates affect lipid metabolism in rats

Linda M. Samuelsson, Wayne Young, Karl Fraser, Gerald W. Tannock, Julian Lee, Nicole C. Roy

Metabolomics (March 2016) DOI 10.1007/s11306-016-1016-7

Abstract

Introduction: Digestion resistant carbohydrates (DRC) are complex carbohydrates that resist digestion and absorption in the small bowel. Diets high in DRC can have wide ranging impacts on the health of the host, which include changes to immunity and allergy, incidence of cardiovascular disease, and obesity.

Objectives: The aim of this study was to characterise the effects of DRC (inulin, konjac or resistant starch) on large intestinal short-chain fatty acid (SCFA) concentrations and serum metabolite and lipid profiles.

Methods: A rat model was used to compare the effects of feeding a basal diet or the basal diet containing 5% inulin, konjac or resistant starch for 14 days.

Results: Of the three DRC, inulin had the greatest effect; ten serum phospholipids differed significantly in abundance between inulin-treated and control rats. In particular phosphatidylcholines and lysophosphatidylcholines containing fatty acyl chains 22:5, 22:4, 20:4, 18:0 and 16:0 were increased in the inulin-fed group, whereas phosphocholines containing fatty acyls 20:5 and 22:6 were decreased.

Conclusion: These results indicated an impact on both n -3 and n -6 fatty acid metabolism as a result of inulin dietary intake. Increased intestinal concentrations of SCFA were detected in rats fed DRC, but only inulin caused appreciable changes to serum lipid profiles.

The people involved

Professor Gerald Tannock is a Principal Investigator in the Department of Microbiology and Immunology. This work was undertaken with other New Zealand researchers from AgResearch, the Riddet Centre of Research Excellence, Plant and Food Research and Gravida National Centre for Growth and Development. 

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February 2016

Sub-clinical detection of gut microbial biomarkers of obesity and type 2 diabetes

Moran Yassour, Mi Young Lim, Hyun Sun Yun, Timothy L. Tickle, Joohon Sung, Yun-Mi Song, Kayoung Lee, Eric A. Franzosa, Xochitl C. Morgan, Dirk Gevers, Eric S. Lander, Ramnik J. Xavier, Bruce W. Birren, GwangPyo Ko and Curtis Huttenhower

Genome Medicine (February 2016) DOI 10.1186/s13073-016-0271-6

Abstract

Background: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression.

Methods: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background.

Results: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities.

Conclusions: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions.

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January 2016

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma

Noelyn A. Hung, Ramona A. Eiholzer, Stenar Kirs, Jean Zhou, Kirsten Ward-Hartstonge, Anna K. Wiles, Chris M. Frampton, Ahmad Taha, Janice A. Royds and Tania L. Slatter

Modern Pathology advance online publication, 15 January 2016; doi:10.1038/modpathol.2015.156

Summary of the work

Using RNA-Seq and immunohistochemistry microscopy, it was determined that a majority of patients with no defined telomere-maintenance mechanism contained high infiltrates of tumour-associated macrophages in Glioblastoma.  Patients with a high infiltrate of tumour-associated macrophages were associated with poorer patient outcomes, suggesting that these may be a potential target for treatment in glioblastoma.  

Abstract

Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumorassociated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P = 0.0004, and telomerase with and without macrophages P = 0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1–3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs 476% for all other subtypes, P = 0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.

The people involved

Kirsten Ward Hartstonge was funded by The Centre for Translational Cancer to complete a Summer Studentship (2013/2014). The project was a collaboration between Dr Roslyn Kemp (Department of Microbiology and Immunology) and Dr Tania Slatter (Department of Pathology). The Gut Health Network funded the consumables. 

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December 2015

Distinct immune signatures in the colon of Crohn's disease and Ankylosing spondylitis patients in the absence of inflammation

Dunn, E.T.J., Taylor, E.S., Stebbings, S., Schultz, M., Butt, A.G. and Kemp, R.A.

Immunology and Cell Biology (2015), 1-9. doi:10.1038/icb.2015.112.

Abstract

Crohn's disease (CD) is an inflammatory bowel disease characterized by patchy inflammation of the gastrointestinal tract. Ankylosing spondylitis (AS) is primarily characterized by inflammation of the lower vertebral column, and many patients with AS present with inflammatory gut symptoms. Genome wide association studies have highlighted significant overlap in short nucleotide polymorphisms for both diseases. We hypothesized that patients with CD and AS have a common intestinal immune signature, characterized by inflammatory T cells, compared with healthy people. We designed a pilot study to determine both the feasibility of defining complex immune signatures from primary tissue, and differences in the local immune signature of people with inflammatory diseases compared with healthy people. Intestinal biopsies were obtained by colonoscopy from healthy patients, non-inflamed regions of CD patients and AS patients with inflammatory gut symptoms. A flow cytometry platform was developed measuring polyfunctional T cell populations based on cytokines, surface molecules and transcription factors. There was overlap in the immune signature of people with CD or AS, characterized by changes in the frequency of regulatory T cells, compared with healthy people. There were significant differences in frequencies of other polyfunctional T cell populations-CD patients had an increased frequency of T cells producing IL-22 and IFNγ, while AS patients had an increased frequency of T cells producing IL-2; compared with healthy people. These data indicate that the local immune signature could be described in these patients and that distinct immune mechanisms may underlie disease progression.

The people involved

Elliott Dunn is a MSc graduate of Ros Kemp's lab, and Edward Taylor is a current PhD student. Simon Stebbings and Michael Schultz are based in the University of Otago's Department of Medicine, and Grant Butt is in the Department of Physiology.


November 2015

Gold nanoparticles decorated with sialic acid terminated bi-antennary N-glycans for the detection of influenza virus at nanomolar concentrations

Vivek Poonthiyil, Prashanth T. Nagesh, Matloob Husain, Vladimir B. Golovko and Antony J. Fairbanks

ChemistryOpen, December 2015, DOI: 10.1002/open.201500109

Summary of the work

This paper describes the discovery that carbohydrate molecules isolated from a cheap source – chicken eggs – and immobilized on gold nanoparticles can be used for the selective detection of influenza virus in a sample. Therefore, this finding has the potential for developing a cheaper diagnostic method for detecting influenza virus.

The associated artwork was chosen for the cover of this issue of ChemistryOpen. The cover picture shows the isolation of complex bi-antennary oligosaccharides from hens’ eggs and their conjugation to gold nanoparticles. Gold nanoparticles carrying these sugars can then bind to specific receptors (hemagglutinin) on the surface of the influenza virus, causing particle aggregation, which changes their spectroscopic properties. Upon aggregation, they undergo a red-shift in their surface plasmon resonance, as illustrated by the bound particles shining in the cover image. These changes in spectroscopic properties are the basis of a detection system capable of detecting viral hemagglutinin at nanomolar concentrations, as well as the virus itself.

Abstract

Gold nanoparticles decorated with full-length sialic acid terminated complex bi-antennary N-glycans, synthesized with glycans isolated from egg yolk, were used as a sensor for the detection of both recombinant hemagglutinin (HA) and whole influenza A virus particles of the H1N1 subtype. Nanoparticle aggregation was induced by interaction between the sialic acid termini of the glycans attached to gold and the multivalent sialic acid binding sites of HA. Both dynamic light scattering (DLS) and UV/Vis spectroscopy demonstrated the efficiency of the sensor, which could detect viral HA at nanomolar concentrations and revealed a linear relationship between the extent of nanoparticle aggregation and the concentration of HA. UV/Vis studies also showed that these nanoparticles can selectively detect an influenza A virus strain that preferentially binds sialic acid terminated glycans with a(2!6) linkages over a strain that prefers glycans with terminal a(2!3)-linked sialic acids.

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chemistry open cover


October 2015

Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway

Ryan Harvey, Catherine McCaughan, Lyn M. Wise, Andrew A. Mercer, Stephen B. Fleming

Virus Research 208 (2015) 180–188

Summary of the work

Interferons (IFNs) are a multifunctional family of cytokines that play a critical role as a first line of defence against viral infection. Activation of the Janus Kinase/signal transducer and activation of transcription (JAK/STAT) signalling pathway by IFNs leads to the production of numerous IFN stimulated genes (ISGs) that block viral replication. Our findings show that the parapoxvirus, orf virus blocks the expression of ISGs induced by type I and type II IFNs. This is the first report that describes the inhibition of ISG expression by a parapoxvirus. Moreover we have shown that Orf virus has evolved a mechanism to impair the JAK/STAT signalling pathway by dephosphorylating STAT1 in virus-infected cells that does not require new viral gene synthesis.

Abstract

Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type I IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-γ or IFN-α respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-γ or IFN-α treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway.

The people involved

Ryan Harvey completed an MSc under the supervision of Dr Steve Fleming in the Virus Research Unit (VRU), Department of Microbiology and Immunology, in 2014. Catherine McCaughan was a technician in the VRU and retired earlier this year. Dr Lyn Wise is a Research Fellow in the VRU and Professor Andy Mercer is the Director of the VRU. 

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September 2015

Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

Adrian G. Patterson, James T. Chang, Corinda Taylor and Peter C. Fineran

Nucleic Acids Research, 2015, doi: 10.1093/nar/gkv517

Summary of the work

In this paper, the authors show that the expression of the Type I-F CRISPR-Cas system is controlled by CRP-cAMP and GalM. Mutation of the genes encoding these cellular components influences CRISPR-Cas acquisition and interference processes. These findings suggest that a metabolite-sensing regulatory network exists and that perturbation of normal metabolic flux within the cell may trigger CRISPR-Cas activity.

The people involved

Adrian Patterson is a second-year PhD student under the supervision of Associate Professor Peter Fineran. James Chang is a former Msc student of Peter's who is carrying out his PhD under the supervision of Professor Clive Ronson. Corinda Taylor works as a Research Technician in the Fineran Lab. 

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August 2015

Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging

Chris Greening, Carlo R. Carere, Rowena Rushton-Green, Liam K. Harold, Kiel Hards, Matthew C. Taylor, Sergio E. Morales, Matthew B. Stott, and Gregory M. Cook

PNAS, August 18, 2015, vol. 112 no. 33

Summary of the work

The mechanisms that the “dormant microbial majority” use to remain energized in nutrient-starved soil ecosystems have long remained elusive. In this work, we used an isolate of the highly abundant but poorly understood soil phylum Acidobacteria as a model for understanding microbial persistence mechanisms. When the bacterium entered a persistent state due to nutrient-exhaustion, we showed it could scavenge the trace concentrations of molecular hydrogen gas (H2) found in ambient air using a specialized high-affinity enzyme. These findings demonstrate that Acidobacteria can consume H2 and contribute to global hydrogen cycling. We propose that consumption of trace gases such as H2 provides a dependable general mechanism for dominant soil phyla to generate the maintenance energy required for long-term survival.

The people involved

Lead author Chris Greening is a PhD graduate of the Department of Microbiology and Immunology, while Liam Harold and Kiel Hards are current PhD students and Rowena Rushton-Green is an undergraduate student. Sergio Morales and Greg Cook are academic researchers in the department. The work was undertaken with colleagues from CSIRO (Australia) and GNS Science (New Zealand).

Read the media release on the University of Otago website

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Greg Cook and Chris Greening


July 2015

Receptor-mediated exopolysaccharide perception controls bacterial infection

Y. Kawaharada, S. Kelly, M. Wibroe Nielsen, C. T. Hjuler, K. Gysel, A. Muszyński, R. W. Carlson, M. B. Thygesen, N. Sandal, M. H. Asmussen, M. Vinther, S. U. Andersen, L. Krusell, S. Thirup, K. J. Jensen, C. W. Ronson, M. Blaise, S. Radutoiu and J. Stougaard

Nature 523, 308–312 (16 July 2015) doi:10.1038/nature14611

Summary of the work

Clover and other legumes form a unique symbiotic relationship with bacteria known as rhizobia, which they allow to infect their roots. This leads to root nodules being formed in which the bacteria convert nitrogen from the air into ammonia that the plant can use for growth. This study elucidates how legumes perceive and distinguish compatible bacteria based on the exopolysaccharides featuring on the invading cells’ surfaces. The findings have implications for improving the understanding of how other plant and animal species interact with bacteria in their environment and defend themselves against hostile infections.

Abstract

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume–rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3regulates bacterial passage through the plant’s epidermal cell layer. Finally, we show that Epr3expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant–bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

The people involved

This research was the result of a collaboration between the Department of Microbiology and Immunology's Professor Clive Ronson and colleagues in Denmark at the Centre for Carbohydrate Recognition and Signalling (CARB).

Read the media release on the University of Otago website

Read the paper online


June 2015

Reducing the Oxidation Level of Dextran Aldehyde in a Chitosan/Dextran-Based Surgical Hydrogel Increases Biocompatibility and Decreases Antimicrobial Efficacy

Maggie Chan, Heather J. L. Brooks, Stephen C. Moratti, Lyall R. Hanton and Jaydee D. Cabral

International Journal of Molecular Sciences, 16 June 2015

Summary of the work

The results of this study demonstrate the importance of oxidation level in the biocompatibility and antimicrobial efficacy of a surgical hydrogel. Although the hydrogel tested offers reduced antimicrobial ability when compared to the higher dextran aldehyde alternative, it functioned effectively as a physical adhesion barrier when used with dressings. A more moderate level of oxidation or the inclusion of broad-spectrum antimicrobial agents could be investigated in future to combine the benefits of biocompatibility, antimicrobial efficacy and adhesion prevention. This could lead to reduced healing times, lowered infection rates, and eliminate the need for second look surgeries to remove adhesions.

Abstract

A highly oxidized form of a chitosan/dextran-based hydrogel (CD-100) containing 80% oxidized dextran aldehyde (DA-100) was developed as a post-operative aid, and found to significantly prevent adhesion formation in endoscopic sinus surgery (ESS). However, the CD-100 hydrogel showed moderate in vitro cytotoxicity to mammalian cell lines, with the DA-100 found to be the cytotoxic component. In order to extend the use of the hydrogel to abdominal surgeries, reformulation using a lower oxidized DA (DA-25) was pursued. The aim of the present study was to compare the antimicrobial efficacy, in vitro biocompatibility and wound healing capacity of the highly oxidized CD-100 hydrogel with the CD-25 hydrogel. Antimicrobial studies were performed against a range of clinically relevant abdominal microorganisms using the micro-broth dilution method. Biocompatibility testing using human dermal fibroblasts was assessed via a tetrazolium reduction assay (MTT) and a wound healing model. In contrast to the original DA-100 formulation, DA-25 was found to be non-cytotoxic, and showed no overall impairment of cell migration, with wound closure occurring at 72 h. However, the lower oxidation level negatively affected the antimicrobial efficacy of the hydrogel (CD-25). Although the CD-25 hydrogel’s antimicrobial efficacy and anti-fibroblast activity is decreased when compared to the original CD-100 hydrogel formulation, previous in vivo studies show that the CD-25 hydrogel remains an effective, biocompatible barrier agent in the prevention of postoperative adhesions.

The people involved

Maggie Chan is a graduate and former Research Assistant, who worked under the supervision of Dr Heather Brooks from the Department of Microbiology and Immunology. Stephen Moratti, Lyall Hanton and Jadee Cabral are project collaborators from the Department of Chemistry.

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May 2015

Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization

Elaine Chiu, Marcel Hijnen, Richard D. Bunker, Marion Boudes, Chitra Rajendran, Kaheina Aizel, Vincent Oliéric, Clemens Schulze-Briese, Wataru Mitsuhashi, Vivienne Young, Vernon K. Ward, Max Bergoin, Peter Metcalf, and Fasséli Coulibaly

PNAS, March 31, 2015

Summary of the work

In this paper, the authors present the structure of protein crystals that form naturally in virally infected insects and boost the insecticidal activity of oral pathogens. These findings may guide their use as synergetic additives to common bioinsecticides. X-ray crystallography is a powerful approach for understanding the structure and function of biological macromolecules, but has so far been largely limited to molecules that form high-quality crystals in the laboratory.

Abstract

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.

The people involved

Vivienne Young and Professor Ward are based in the Department of Microbiology and Immunology at the University of Otago. Colleagues Elaine Chiu, Richard Bunker and Peter Metcalf are with Auckland University's School of Biological Sciences, with other authors of the paper based in Australia, Switzerland, Japan and France.

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April 2015

Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

David W. Taylor, Yifan Zhu, Raymond H. J. Staals, Jack E. Kornfeld, Akeo Shinkai, John van der Oost, Eva Nogales, Jennifer A. Doudna

Science, April 2015.

Summary of the work

In the paper, the authors show how CRISPR-Cas, a surveillance complex in the bacterial immune system, is able to target specific sites on RNA molecules to destroy invading viruses and other foreign genetic elements. The finding could lead to the development of tailor-made RNA-editing tools.

Abstract

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. While Type I and Type II CRISPR-Cas surveillance complexes target double-stranded DNA, Type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy (cryo-EM) reconstructions of native Type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumb-like β-hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nt intervals. The Cmr complex is architecturally similar to the Type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.

The people involved

Dr Raymond Staals is a Postdoctoral Fellow in the Department of Microbiology and Immunology at the University of Otago. The work was undertaken in collaboration with colleagues at his home university in Wageningen, Netherlands, along with The University of California, Berkeley.

Read the full paper online

Read the media release on the University of Otago website


March 2015

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

Joshua P. Ramsay, Laura G. L. Tester, Anthony S. Major, John T. Sullivan, Christina D. Edgar, Torsten Kleffmann, Jackson R. Patterson-House, Drew A. Hall, Warren P. Tate, Michael F. Hynes, and Clive W. Ronson

PNAS 112 (13), pp 4104–4109.

Summary of the work

Integrative and conjugative elements (ICEs) facilitate horizontal transfer of multiple genetic determinants. Here we show that a programmed ribosomal frameshift (PRF) contributes to the regulation of ICE transfer. The low-frequency PRF fuses the coding sequences of two genes, resulting in a single-protein Frameshifted excision activator (FseA) that activates ICE excision. An antiactivator, QseM, known to disrupt the quorum-sensing regulator TraR, also disrupted FseA. The evolved PRF site, together with the dual-target antiactivator, QseM, likely provides robust suppression of ICE transfer in the face of the inherent biological noise of quorum-sensing autoinduction. This work illustrates how a complex multipartite regulatory system has assembled through evolution to form a robust genetic toggle to control gene transcription and translation at both single-cell and cell-population levels.

The people involved

Dr Josh Ramsay was a Health Sciences Career Development Postdoctoral Fellow in Prof Clive Ronson’s lab for the majority of this work; he is now a lecturer at Curtin University in Perth, WA. Laura Tester and Anthony Major were Masters students in Microbiology jointly supervised by Josh and Clive, Dr John Sullivan is an Assistant Research Fellow in Clive’s lab; Tina Edgar, Dr Torsten Kleffmann and Prof Warren Tate are collaborators in the Department of Biochemistry at Otago, Jackson Patterson-House and Drew Hall were Honours students with Josh at Curtin University, Prof Michael Hynes was a Visiting Professor in Clive’s lab from the University of Calgary in Canada.

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February 2015

Biogeography and biophysicochemical traits link N2O emissions, N2O emission potential and microbial communities across New Zealand pasture soils

Sergio E. Morales, Neha Jha, and Surinder Saggar

Soil Biology & Biochemistry, 82 (2015), 87e98

Summary of the work

The results of this study indicate that, like plants and animals, microbial communities controlling emissions of the greenhouse gas nitrous oxide (N2O) are influenced by latitude. Although the mechanism is unclear, it suggests that soils closer to the poles have a higher, untapped potential for emitting greenhouse gases than those closer to the Equator. Under changing global conditions, this could result in higher gas emissions if temperature and moisture patterns shift.

The people involved

Dr Sergio E. Morales is a Lecturer in the Department of Microbiology and Immunology. This work was undertaken in collaboration with Landcare Research and Massey University, part of the New Zealand Agricultural Greenhouse Gas Research Centre.

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January 2015

A high-throughput screening assay for identification of inhibitors of the A1AO-ATP synthase of the rumen methanogen Methanobrevibacter ruminantium M1

Htin Lin Aung, Debjit Dey, Peter H. Janssen, Ron S. Ronimus, Gregory M. Cook

Journal of Microbiological Methods, Volume 110, March 2015, Pages 15–17
doi:10.1016/j.mimet.2014.12.022

Summary of the work

The high-throughput screen developed in this study provides a biochemical platform to accelerate discovery of novel inhibitors of rumen methanogens to mitigate methane emissions.

The people involved

Dr Htin Lin Aung is a Postdoctoral Fellow in Professor Gregory Cook's laboratory. This work was undertaken in collaboration with AgResearch, part of the Pastoral Greenhouse Gas Research Consortium. The PGgRc research programme aims to provide New Zealand livestock farmers with the knowledge and tools to mitigate greenhouse gas emissions from the agricultural sector.

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November 2014

Mannosylation of Virus-Like Particles Enhances Internalization by Antigen Presenting Cells

Farah Al-Barwani, Sarah L. Young, Margaret A. Baird, David S. Larsen and Vernon K. Ward

PLoS ONE 9(8): e104523
doi:10.1371/journal.pone.0104523

Abstract

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.

The people involved

Farah Al-Barwani is a PhD candidate in the Department of Microbiology and Immunology, working under the supervision of Professor Vernon Ward. The work was undertaken in collaboration with Sarah Young and Margaret Baird of the Department of Pathology and David Larsen from the Department of Chemistry.

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October 2014

Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach

Charlotte M. Wilson, Diane Loach, Blair Lawley, Tracey Bell, Ian M. Sims, Paul W. O'Toole, Aldert Zomer and Gerald W. Tannock

Applied and Environmental Microbiology, 2014, 80(19):6104
doi:10.1128/AEM.01876-14

Summary of the work

Habitat studies that measure bacterial functions can reveal mechanisms of ecological success of gut commensals, and their impact on host physiology. The study by Wilson et al. (AEM01876-14) showed that, while urease gene transcription was upregulated in vivo by L. reuteri as an acid tolerance mechanism, urea was metabolized to a greater extent in Lactobacillus-free mice compared to animals colonized by strain 100-23. This was because fecal-type bacteria in the stomach of Lactobacillus-free mice had high urease activity, but were suppressed by L. reuteri. This unexpected impact of lactobacilli on global urea hydrolysis highlighted the role of gut commensals on mammalian nitrogen recycling.

The people involved

The article is based on some transcriptome experiments conducted by PhD student Charlotte Wilson, which led to further bacteriological and DNA-based studies, as well as collaborative work with chemists Drs Tracey Bell and Ian Sims at Callaghan Innovation. The article was selected by the editors of Applied and Environmental Microbiology for inclusion in "Spotlight," a feature in the Journal highlighting research articles in the upcoming issue that have been deemed of significant interest and includes short descriptions of four to six especially meritorious articles (reproduced below). The research was supported by the Marsden Fund.

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September 2014

A new host protein that inhibits influenza A Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate acetylated microtubules

Matloob Husain and Chen-Yi Cheung

Journal of Virology (2014)
doi:10.1128/JVI.00727-14

Summary of the work

Dr Matloob Husain

Host cells produce many proteins that have the natural ability to restrict influenza A virus infection. In this paper, we have described a new class of host protein, histone deacetylase 6 (HDAC6) that inhibits the influenza A virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza A virus assembly via its substrate acetylated microtubules. HDAC6 is a multi-substrate enzyme and regulates multiple pathways in the host cell, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of wide range of diseases. Influenza A virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza A strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza potential of endogenous HDAC6 to inhibit influenza A virus infection.

The people involved

The research was conducted by Dr Matloob Husain and his technician Chen-Yi Cheung over the last two and half years, and supported by the Health Research Council, Otago School of Medical Sciences, and Department of Microbiology and Immunology.

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August 2014

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer

*Richter, C., *Dy, R.L., *McKenzie, R.E., Watson, B.N.J., Taylor, C., Chang, J.T., McNeil, M.B., Staals, R.H.J. and Fineran, P.C.
*equal contributions

Nucleic Acids Research (2014)
doi: 10.1093/nar/gku527
Journal impact factor = 8.28

Summary of the work

The interactions between bacteria and their ‘parasites’, such as viruses and plasmids, underpin global nutrient cycles, the evolution of pathogens and antibiotic resistance. Bacteria and archaea protect themselves using an adaptive immune system, termed CRISPR-Cas, which has a sequence-specific genetic memory of previous invaders. This memory produces short interfering RNAs that specifically target and destroy invaders. Recently, CRISPR-Cas systems have revolutionised precision genome editing and have, for example, enabled the correction of genetic defects in adult mice. Despite this stunning technological advance, fundamental knowledge is lacking about how memories are derived from invaders. In this new study we show that the memory formation process is capable of rapidly eliciting new protective memories when facing invaders that were mutated following previous encounters. Exactly how these memories with partial recognition stimulate new memory formation is unknown, but our data have led us to propose a bi-directional translocation model for acquisition of new memories. Understanding these systems has broad implications for biotechnology and prokaryotic evolution.

The people involved

The research was conducted by members of Peter Fineran’s lab over the last 2 years. Much of the work was the result of a recent Honours student (Becca McKenzie), a post-doc (Ron Dy) and a PhD student (Corinna Richter). Other authors include summer and MSc students, post-docs and a technician. The major support was from a Rutherford Discovery Fellowship and the University of Otago.

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July 2014

NK Cells Are Required for Dendritic Cell–Based Immunotherapy at the Time of Tumor Challenge

Anthea L. Bouwer, Sarah C. Saunderson, Felicity J. Caldwell, Tanvi T. Damani, Simon J. Pelham, Amy C. Dunn, Ralph W. Jack, Patrizia Stoitzner and Alexander D. McLellan

Journal of Immunology. 2014 Mar 1;192(5):2514-21.
doi: 10.4049/jimmunol.1202797
Journal impact factor: 5.5

Summary of the work

Recent research conducted by Associate Professor Alex McLellan and colleagues shows that bacteria may assist the body’s immune system response against cancer cells and help fight tumours like melanoma.

The article published in the official journal of the American Association of Immunologists, the Journal of Immunology, demonstrates that bacteria stimulate a type of immune response that results in more effective killer cell attacks against cancer.

Read the press release on the University of Otago website

Abstract

Increasing evidence suggests that NK cells act to promote effective T cell–based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria–dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Read the full paper online